Steady-state and pre-steady-state kinetics of the trypsin-catalysed hydrolysis of alpha-CBZ-L-lysine-p-nitrophenyl ester.
The catalytic properties of bovine tyrpsin (EC 3.4.21.4) have been investigated using a synthetic chromogenic substrate: alpha-CBZ-L-lysine-p-nitrophenyl ester (ZLNPE). The use of ZLNPE allows the determination of trypsin down to a concentration of 2 . 10(-9) M. Steady-state and pre-steady-state data have been in the framework of the minimum three-step mechanism: (Formula: see text). The pH-dependence of the kinetic parameters shows that at acid pH values (congruent to 2.6) the k+3 step is rate limiting in catalysis, whereas for pH values higher than 4.8 k+2 becomes rate limiting. This change in rate-limiting step with pH illustrates the danger in the assumption that kcat vs. pH profiles for protease action on substrates with good leaving groups are equivalent to k+3 vs. pH profiles.
D000818:Animals; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism*
C024992:N(alpha)-carbobenzoxylysine-4-nitroanilide; D014357:Trypsin; D008239:Lysine